PT99063B - PROCESS FOR THE PRODUCTION OF A GLYCOPROTEIN AND VACCINES THAT CONTAINS IT - Google Patents

Abstract

This invention provides novel, substantially uncleavable forms of gp 160, and vaccine formulations containing gp 160 or a derivative thereof, adjuvanted with 3-D Mpl. The compositions are useful for the immunotherapeutic and immunoprophylatic treatment of HIV infections.

Description

DESCRIPTIVE MEMORY

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The present invention relates to a process for the production of novel, substantially non-cleavable forms of a 160 KD molecular weight (gp 160) glycoprotein, and vaccine formulations containing gp 160 or a derivative thereof, having monophosphoryl-lipid A as an adjuvant. 3-deacylated (3D MPL). The compositions are useful for the immunotherapeutic and immunoprophylactic treatment of HIV infections.

More specifically, the present invention relates essentially to a process for the production of a modified HIV protein gpl60 in which amino acids other than lysine or arginine are provided at positions 502 and 510 independently, which process consists of the expression of a DNA sequence which encodes said protein in a recombinant eukaryotic host.

This invention relates to new vaccines and new pharmaceutical formulations and to their production and use in the treatment of AIDS. In particular, the invention relates to the use of gp10 or its derivatives, including new forms of gp 160 in a vaccine, having as adjuvant monophosphoryl-lipid A 3-deacylated.

Retroviruses, that is, viruses of the Retroviridae family, constitute a large family of coated, icosahedral viruses of about 150 nm having a nucleocapsid wrapped inside the nucleus structure and having RNA as genetic material. The family comprises oncoviruses such as sarcoma and leukemia viruses, certain immunodeficiency viruses and lentiviruses.

The human immunodeficiency virus type 1 (HIV-1) is the etiological agent of the acquired immunodeficiency syndrome, also known as AIDS. This retrovirus has a complex genetic organization, including long terminal repeats (LTRs), the gag, pol and env genes, and other genes. This retrovirus carries a number of antigens that are potential candidates either alone or in concert as vaccine agents capable of inducing a protective immune response.

The HIV-1 envelope protein is synthesized as a precursor to the polyprotein which is subsequently glycosylated within the infected cells to give rise to a glycoprotein with a molecular weight of 160 kDa (gp 160), which is processed by proteolysis into an external glycoprotein gp 120 and transmembrane protein gp 41.

A region encoding DNA for proviral HIV can be prepared from any of the genomic clones of the immunodeficiency virus referred to in the literature. See, for example, Shaw et al., Science 226: 1165 (1984); Kramer et al., Science 231: 1580 (1986). Alternatively, an immunodeficiency virus genomic clone can be prepared from viruses isolated from clinical specimens using standardized DNA cloning techniques. See, for example, Gallo et al. , U.S. Patent No. 4,520-113; Montagnier et al., U.S. Patent No. 4,708,818.

The HIV-BH10-2 proviral DNA sequence is described by Ratner et al., Human Retroviruses and AIDS 1989, HIV Sequence Database ed. Gerald Meyers, Los Alamos National Laboratory. The sequence is 8932 bp in length, with the env gene located at 5580-8150 and its cleavage sites at 7088 and 7112 corresponding to amino acids 503 and 511 (cleavage after these residues) (numbering according to Los base data Alamos).

In accordance with the present invention, HIV gp 160 is provided which has been modified to provide amino acids other than lysine or arginine at positions 502 and 510 independently.

Suitable substitutions for lysine are those that resemble lysine in hydrophilicity and size, such as histidine, threonine, serine, asparagine, aspartic acid, glutamine and glutamic acid. Of these, the preferred amino acid is glutamic acid. The preferred protein is both non-cleavable due to the mutations introduced and also capable of producing cross-neutralizing antibody, which depends on the correct flexibility of the protein.

More preferably, the invention provides the modified protein of the invention in an oligomeric form. In particular with a relative molecular weight of 640 kDa. Based on the molecular weight of monomer gp 160, this form is thought to be tetrameric. This is advantageous since viral surface proteins naturally exist as oligomers which form in-vivo spikes that protrude from the viral surface. As neutralization epitopes are conformational, it is clearly important to mimic as far as possible the naturally occurring form of the antigen, as this will provide a more relevant immune response.

In a preferred presentation the invention provides the modified protein of the invention, in an oligomeric and substantially pure form.

Substantially pure means at least 75% purity, more preferably 90% purity, most preferably 99% purity.

In another aspect, the invention provides a process for the preparation of modified HIV gp 160 according to the invention, which process consists of expressing DNA encoding said modified protein in a recombinant eukaryotic host cell and recovering the modified protein product.

DNA polymer comprising a nucleotide sequence encoding the modified protein is also part of the invention.

The recombinant DNA molecule of the invention can be prepared according to the invention by condensing appropriate mono-, di- or oligomeric nucleotide units.

The preparation can be carried out chemically, enzymatically, or by combining the two methods, in vitro or in vivo as appropriate. Thus, the DNA molecule can be prepared by enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D. M. Roberts et al in Biochemistry 1985, 24, 5090-5098.

DNA fragments can be obtained by digesting DNA containing the required nucleotide sequences with appropriate restriction enzymes, by chemical synthesis, by enzymatic polymerization, or by a combination of these methods.

Digestion with restriction enzymes can be performed in an appropriate buffer at a temperature between 20 ° -70 ° C, usually in a volume of 50 μΐ or less with 0.1-10 pg of DNA.

Enzymatic polymerization of DNA can be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dGTP, dGTP and dTTP as required at a temperature of 10 ° - 37 ° C, usually in a volume of 50 μΐ or less. The fragments can be polymerized and amplified by polymerase chain reaction using Taq polymerase (ref. PCR Protocols 1989 - a guide for Methods and Applications, Ed. M.A. Innis et al., Academic Press).

Enzymatic ligation of DNA fragments can be performed using DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4 ° C at room temperature, usually in a volume of 50 μ or less.

in other publications Matthes, M. Singh,

Chemical synthesis of the DNA molecule or fragments can be performed by conventional chemical processes with phosphotriester, phosphite or phosphoramidite, using solid phase techniques such as those described in Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual (ed. HG Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or scientific, for example MJ Gait, HWD

B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24. 5771; M.D. Matteucci and M.H. Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D. Matteucci and M.H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S.P. Adams et al., Journal of the American Chemical Society, 1983, 105, 661; N.D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W.D. Matthes et al .. EMBO Journal, 1984, 3, 801. Preferably, an automatic DNA synthesizer is used.

DNA polymer encoding the modified protein can be prepared by mutagenesis directed at the cDNA site encoding the unmodified protein, by conventional methods such as those described by G. Winter et al in Nature 1982, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6500.

The invention also extends to a vector comprising the recombinant DNA molecule of the invention and to a recombinant vaccine virus containing said vector.

vector can be prepared according to the invention by cleaving a vector to provide a linear segment of DNA having intact replication, and by linking said linear segment and one or more DNA molecules, which together with said linear segment complete the recombinant DNA molecule of the invention.

The recombinant host cell of the invention can be prepared by transforming a vaccine virus with a vector of the invention.

modified protein product is isolated from conditioned medium by standard protein isolation and purification techniques. Detergents, for example, Decyl PEG-300, DO Decyl PEG Triton X100. Thesit Deoxycholate, can be added in order to lyse the cell and release the modified protein from the cell membrane material. Of these, Decyl PEG-300 and Thesit Deoxycholate are preferred, and the Decyl Triton X100 Modified DO protein can then be purified by a series of ultrafiltration steps, ultracentrifugation steps, selective precipitations with, for example, ammonium sulfate or PEG, centrifugation with gradient of density in CsCl or sucrose or metrizamide gradients and / or chromatographic steps, such as affinity chromatography, immunoaffinity chromatography, HPLC, reverse phase HPLC, cation and anion exchange, size exclusion chromatography, preparative isoelectric focus is preferred. Purification using immunoaffinity chromatography is preferred. During or following purification, the modified protein can be treated with, for example, formaldehyde, glutaraldehyde or NAE to improve stability or immunogenicity.

For the preparation of the oligomeric form of the invention it is preferred to use mild conditions during the purification steps. In particular, the reducing agent should be avoided, and non-ionic detergents such as Decyl PEG-300 (polyethylene glycol monodecylether 300) are preferred over ionic detergents for cell lysis and solubilization during the chromatographic steps. A preferred affinity chromatographic medium is Lentil lectin Sepharose and a preferred immunoaffinity chromatography medium is an anti-gp 160 monoclonal antibody such as 178.1 (WO 90/06358) coupled to an appropriate vehicle such as Trisacryl | (IBF) activated with glutaraldehyde.

The modified protein can be adsorbed from the monoclonal antibody in the presence of the octyl detergent glucopyranoside which can be removed by dialysis. Detergents are preferably used in concentrations above their theoretical critical micellar concentration.

The modified protein produced in accordance with this invention is useful as a diagnostic agent for detecting exposure to HIV. The modified protein is also useful in vaccines to prevent infection or to inhibit or prevent disease progression.

This invention also relates to a vaccine and pharmaceutical compositions containing the modified protein of this invention. Such compositions will contain an immunoprotective amount of the modified protein of this invention and can be prepared by conventional techniques.

In the vaccine of the invention, an aqueous solution of the protein can be used directly. Alternatively, the protein, with or without prior lyophilization, can be mixed or absorbed with any of several known adjuvants. Such adjuvants include, but are not limited to, aluminum hydroxide,. muramyl dipeptide and saponins such as Quil A, 3D-MPL (3-deacylated monophosphoryl lipid A), or TDM. As yet another alternative example, the protein can be encapsulated in microparticles such as liposomes. In yet another alternative example, the protein can be conjugated to an immunostimulating macromolecule, such as dead Bordetella or tetanus toxoid.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, USA 1078. Encapsulation within liposomes is described, for example, by Fullerton, US Patent. USA No. 4,235,877. Conjugation of proteins into macromolecules is disclosed, for example, by Likhite, U.S. Patent No. 4,372,945 and by Armor et al., U.S. Patent No. 4,474,757.

The amount of modified protein of the present invention in each dose of vaccine is selected as an amount that induces an immunoprotective response without significant adverse side effects in typical vaccines. That amount will vary depending on which specific immunogen is used and whether whether or not the vaccine has adjuvants. Generally, it is expected that each dose will comprise 1-1,000 mg of modified protein, preferably 10-200 mg. An optimum amount for a given vaccine can be verified by standard studies involving observation of antibody titers and other responses in individuals. Following initial vaccination, individuals will preferably receive a booster after about 4 weeks, followed by repeated boosters every six months as long as there is a risk of infection.

The invention further provides modified protein of the invention for use in vaccinating a host and use of the modified protein in the preparation of a vaccine.

In addition to vaccination of individuals susceptible to HIV infections, the pharmaceutical compositions of the present invention can be used to treat, immunotherapeutically, patients suffering from HIV infections.

Accordingly, in one aspect of the present invention there is provided a method for treating a human being susceptible to or suffering from HIV infection by administering an effective amount of modified gp 160 as described herein.

Coincidentally with the concept of using sub-unit components produced, for example, by means of recombinant DNA technology, there is a need for adjuvants and / or vehicles to effectively present the immunogens to the host's immune system so that the two arms immune response (antibody neutralization and cell-mediated immunity (DTH)) are produced. In the context of the present invention (i.e. in the prophylactic or therapeutic treatment of HIV infections) we have found that an immunostimulating portion, 3D MPL is capable of stimulating both arms of the immune system.

Accordingly, in a preferred aspect of the present invention, a pharmaceutical formulation comprising gp 160 or an immunological derivative and monosphosphoryl-lipid A (3D-MPL) is provided with an appropriate vehicle.

In a preferred embodiment of the present invention, gp 160 or an immunological derivative thereof and 3D-MPL are presented in an oil-in-water emulsion. This system provides an improved neutralizing activity.

Accordingly, in a preferred aspect of the present invention there is provided a pharmaceutical or vaccine formulation comprising gp 160 or an immunological derivative, 3D-MPL in an oil-in-water vehicle, said vehicle comprising a tetrapoliol emulsion and a non-toxic mineral oil in a solution | buffered saline.

Preferably the vehicle comprises a Pluronic polyol such as Pluronic L121, and squalene or squalene or other metabolizable oils. An emulsifier such as Tween 80 or Tween 28 is preferably provided in order to stabilize the emulsion.

The vehicle preferably contains only submicron particles between 100 and 400 nm.

The concentration of antigen in the final formulation preferably ranges between 10 µg and 150 µg / ml, more preferably between 20 µg and 100 µg / ml.

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The concentration range of the adjuvant, 30-MPL. in the vaccine, it preferably ranges between 10 pg and 100 μgml, more preferably between 25 and 50 gg / ml.

the present invention further provides the vaccine formulations as described herein for use in medicine, in particular for use in the treatment by immunotherapy and prophylactic treatment of HIV infections such as AIDS or AIDS-related complex (ARC).

In another aspect of the present invention, there is provided a method for producing the vaccine comprising gp 160 or an immunological derivative thereof, monophosphoryl lipid A 3D with an appropriate vehicle, the method comprising mixing gp 160 or an immunological derivative thereof with the said vehicle and with monophosphoryl lipid A 3D.

In a presentation, a method is provided for producing a vaccine as described herein in an oil-in-water vehicle in which an oil-in-water emulsion is microfluidized to provide micron particles in said emulsion and mixed with gp 160 or an immune and 3D derivative thereof MPL.

Typically 3D-MPL is pre-mixed with the emulsion, and then the antigen is mixed into the resulting composition.

3D-MPL can be obtained by the methods indicated in British Patent 2,211,502.

Suitable vehicles in this context comprise oil-in-water emulsions. The formulations of the present invention provide improved neutralization titers when compared to conventional vaccine formulations comprising only alum as the adjuvant (the only adjuvant authorized for human use).

The term immunological derivatives is used herein to include immunogenic fragments of gp 160 which when adjuvanted with Monophosphoryl-lipid A 3D are able to raise neutralizing antibodies against HIV-1. As such, for example, the HIV-1 outer membrane glycoprotein gp 120, modified gp 160 as described herein will be included as well as naturally occurring isolated gp 160. Particularly preferred are

derivatives that are also capable of increasing the DTH response.

Thus, with the greatest preference the invention provides the modified form of the protein in oligomeric form which, when purified in mild, non-reducing conditions, is found to have an apparent molecular weight ranging between 600-700 KDa 640 Kd thinking it to be a tetramer. This tetramer can be destabilized when the SDS gel protein is used in non-reducing conditions, thus obtaining a 330 kd dimer and a monomer. The dimer. It may also be reduced under standard reducing conditions to yield the ^F monomer.

All of these forms of gp 160 can be used in the formulations of the present invention.

The production of gp 160 or its derivatives can be carried out by methods known in the art. Typically this will involve the cloning and expression of the gene encoding gp 160 in an appropriate host. The production of recombinate gp 160 (Rgp 160) in this way can be accomplished using the techniques described in Maniatis et al; Molecular Cloning - A Laboratory Manual; Cold Spring Harbor 1982.

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A number of eukaryotic cells and expression systems are available for expression of recombinant DNA molecules. Among these, the most commonly used are yeast, insect or mammalian systems, although the invention is not limited to their use. Such systems use a recombinant DNA molecule of the invention, optionally a selection marker, in some cases, maintenance functions such as a starting point for replication.

Insect cells that can be used in the invention include Drosophila cells and Lepidoptera cells. Useful Drosophila cells include Sl, S2, S3, KC-0 and D. Hydei cells. See, for example, Schneider et al., J. Embryol. Exp. Morph. 27.:353 (1972); Schultz et al., Proc. Natl. Acad. Know. USA 83: 9428 (1986); Sinclair et al., Mol. Cell. Biol. 5: 3208 (1985). Drosophila cells are transinfected by standard techniques, including calcium phosphate precipitation, cell fusion, electroporation and viral transinfection. Cells are grown according to standard cell culture processes in a number of nutrient media, including, for example, M3 media that consist of balanced salts and essential amino acids. See, Lindquist, DIS 58: 163 (1982).

Promoters deemed useful in Drosophila include promoters from mammalian cells such as SV40 as well as promoters from Drosophila, the latter being preferred. Examples of useful Drosophila promoters include the Drosophilla metallothionein promoter, the 70 kilodalton heat shock protein (HSP70) promoter and COPIA LTR. See, for example, DiNocera et al., Proc. Natl. Acad. Know. USA 80; 7095 (1983); McGarry et al., Cell 42: 903 (1985).

Useful Lepidoptera cells include cells from Trichoplusia ni, Spodoptera frucriperda. Heliothis zea, Autographica californica. Rachiplusis or. Galleria melonella. Manduca sixth or other cells that can be infected with Baculovirus, including nuclear polyhedrosis virus (NPV), single nucleocapsid viruses (SNPV9 and multiple nucleocapsid viruses (MNPV). The preferred Baculoviruses are Baculovirus NPV or MNPV because they contain the polyhedrin gene which is highly expressed in infected cells. Particularly exemplified here is the MNPV virus from Autographica californica (AcMNPV). However, other MNPV and NPV viruses can also be used the silkworm virus, Bombyx mori. Lepidoptera cells are co-transinfected with DNA comprising the recombinant DNA molecule of the invention and with the DNA of an infectious Baculovirus by means of standardized transinfection techniques, as discussed above. The cells are cultured according to culture techniques of standardized cells in a range of nutrients, including, for example, TC100 (Gibco Europe; Gardiner et al., J. Inverteb. Pathol. 25.:363 (1975) supplemented with 10% fetal calf serum (FCS). See, Miller et al., In Setlow et al., Eds., Genetic Encrineer ince Principies and Methods. . Volume 8, New York, Plenum, 1986, pages 277-298.

Promoters for use in Lepidoptera cells include promoters from a baculovirus genome. The polyhedrin gene promoter is preferred because the polyhedrin protein is naturally overexpressed over other Baculovirus proteins. The polyhedrin gene promoter from the AcMNPV virus is preferred. See, Summers et al., TAES Bull. NR 1555, May 1987; Smith et al., EP-A-127,839; Smith et al., Proc. Natl. Acad. Know. USA 82: 8404 (1985); and Cochran, EP-A-228,036.

For expression in mammalian cells, the recombinant DNA molecule is also cloned with a cloning vector and is then used for transinfection into mammalian cells. The vector preferably comprises additional DNA functions for gene amplification, for example, a cassette for DHFR expression, and can also comprise additional functions for selection and / or amplification, for example, a neomycin resistance cassette for G418 selection . Other functions can also be used, such as improving transcription. Other functions for episomal maintenance can also be included in the vector

stable, if desired, such as Bovine Papilloma Virus maintenance functions. Alternatively and preferably the cloning vector is a recombinant mammalian virus such as a vaccinia virus.

The vaccinia virus is a particularly useful vector in that recombinants can be rapidly constructed by integrating the foreign gene into a nonessential region of vaccinia DNA thereby retaining infectivity. When properly engineered, proteins are synthesized, processed and transported to the membrane of infected cells. Although vaccinia virus infection leads to cell death, there is little lysis and most cells remain intact, allowing easy extraction of the required protein from infected cells. A vaccinia virus expression system was developed by Barrett et al., Aids Research and Human Retroviruses 1989; 5: 159-171.

Useful mammalian cells include cells from Chinese hake ovary (CHO), NIH3T3, COS-7, CV-I. BHK-21, mouse or rat myeloma, HAK, Vero, HeLa, human diploid cells such as MRC-5 and W138, or chicken lymphoma cell lines, with CV-I and BHK-21 being preferred.

Transinfection and cell culture are performed using standardized techniques. Production in mammalian cells can also be performed by expression in transgenic animals.

Regulatory sequences useful for conducting gene expression in mammalian cell lines or primary mammalian cells include the SV 40 early and late gene promoters, the metallothionein promoter, viral LTRs such as Sarcoma Rous LTR, the Moloney sarcoma virus ( MSV) or the mouse mammalian tumor virus LTR (MMTV), or the major adenovirus late promoter and hybrid promoters such as hybrid BK virus and adenovirus major late promoter. The control element region can also comprise downstream functions, such as regions for polyadenylation, or other functions, such as transcription-enhancing sequences.

Yeasts that can be used in the practice of the invention include those of the Hanensula genera. Pichia, Kluveromyces. Schizosaccharomyces, Candida and Saccharomyces. Useful promoters include the inducible copper promoter (CUP1), promoters of glycolytic genes, for example, TDH3, PGK and ADH, and the promoters PHO5 and ARG3. See, for example, Miyanohara et al., Proc. Natl. Acad. Know. USA 80: 1 (1983); mellor et al., Gene 24: 1 (1983); Hitzeman et al., Science 219: 620 (1983); Cabezon et al., Proc. Natl. Acad. Know. USA 81: 6594 (1984).

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Example 1 (i) Expression of modified gp 160 in CV-I cells

The env HIV gene of the molecular clone BH10 was mutagenized in order to abolish cleavage of the precursor.

For this, the cleavage sequences lis ala lis arg and arg glu lis arg present in these sequences were modified by site-directed mutagenesis for lis / arg x glu arg.

cleavage site 1 (position 502) cleavage site 2 (position 510)

Clone

BH10

AAG GCA AAG AGA AGA GTG GTG LIS ALA LIS ARG ARG VAL VAL

CAG AGA GAA AAA AGA GLN ARG GLU LIS ARG has undergone V V mutation for: AAG GCA GAG AGA AGA GTG GTG CAG AGA GAA GAA AGA

LIS ALA GLU ARG ARG VAL VAL GLN ARG GLU GLU ARG>

The complete mutagenized env gene (nucleotides 5802-8478) was cloned into the vaccinia plasmid transfer vector pGS20 [Mackett MG, Smith L. and Moss B. (1984) J. Virology 49: 857-864] and transferred to vaccinia virus infectious by recombination. Recombinant vaccinia plaques were analyzed for env expression by EIA capture and positive plaques recloned twice in CV-1 cells. The production of gp 160 not cleaved in these cell lines was also verified by RIPA after metabolic labeling of cells infected with recombinant vaccinia. Cell surface expression was confirmed

by fluorescent labeling of the intact cell surface using anti-gp 120 antibodies.

(ii) Purification of modified 160 gp from anchorage-dependent cells

The HIV gp 160 HIV wrapper protein from (i) was purified in a three-step protocol: lysis of host cells and extraction of the antigen with the aid of a detergent following two affinity chromatographic steps. All purification steps were carried out at 4 ° C.

Step 1: Lysis of CV-1 cells and extraction of qp 160 antigen

After thawing, the cell suspension and microcarrier beads corresponding to 20 l of culture were centrifuged at 2,200 xg for 15 min and the floating product was eliminated. The precipitate was resuspended in 1 l of tris buffer / 30 mM HCl pH 8 supplemented with 150 mM NaCl, 1% polyethylene glycol 300 monodecylether (Decyl PEG) and 20 mcg / ml aprotinin. The cells were lysed for 1 hour on ice with occasional manual agitation and centrifuged at 11,300 xg for 20 minutes. After separating the floating product, the pill was washed with 400 ml of lysis buffer and centrifuged at 11,300 xg for 20 minutes. Cell debris and microcarrier beads were eliminated and the combined floating products were used for further purification.

Step 2: Affinity chromatography on Lysed Lentil 4B Sepharose lectin was chromatographed on a balanced Lentil 4B Sepharose lectin (Pharmacia-LKB) column (2.5 cm x 20 cm)

with 30 mM Tris / HCl pH 8 buffer supplemented with 150 mM NaCl and 0.1% PEG Decyl. After supplying the lysate at a flow rate of 1 ml / min, the column was washed with 30 mM Tris / HCl pH 8 buffer supplemented with IM PEG Decyl 0.1% NaCl. Subsequently, the antigen was eluted from the column with 0.5 M methyl α-D-mannopyanoside in equilibration buffer and positive fractions of 160 gp were pooled. The washing and elution steps were performed at a flow rate of 5.5 ml / min.

Step 3: 178.1-Trisacryl immunoaffinity chromatography

Anti-pg 160 monoclonal antibody 178.1 (Patent Publication No. WO 90/06358) was purified from ascitic liquid on a Protein G-Sepharose column (Pharmacia-LKB) and subsequently coupled to glutaraldehyde-activated Trisacryl (IBF) according to the manufacturer's directives. The antibody, which is directed against an epitope on the gp 120 portion of the antigen (loop V), was coupled to a gel density of 1.5 mg / ml.

The resin was packed in a column (2.5 cm x 10 cm) and equilibrated in 30 mM Tris / HCl pH 8 buffer supplemented with 150 mM NaCl and 0.1% PEG Decyl.

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The product eluted in Sepharose lectin Lentil 4B was supplied to a column by overnight recycling at 1 ml / min. Subsequently, the column was washed at a flow rate of 3.3 ml / min with 20 column volumes of 0 mM Tris / HCl pH 8 supplemented with 1 M NaCl and 1% n.octyl β-D-glucopyranoside (OGP). Finally, the antigen was eluted at the same flow rate with 0.1 M citric acid buffer pH 3.3 supplemented with 1% OGP. The elution fractions were neutralized immediately with 1 M Tris / HCl pH 8.8 and the positive antigen fractions were pooled.

(iii) Protein determination

Protein concentrations were determined by the Bradford method (Bradford, 1976) using bovine serum albumin as the standard.

(iv) Antigen determination

The amount of gp 160 antigen was measured by a sandwich ELISA developed in-house using sheep anti-gp 41 as the capture monoclonal antibody and murine anti-gp 120 as the indicator monoclonal antibody. Further detection was performed with a classic biotinylated anti-mouse antibody, streptavidin system and peroxidase.

(v) Electrophoresis in polyacrylamide gel and Western stain

SDS-slab gel electrophoresis was performed on 10% polyacrylamide gels according to the Laemmli method (Laemmli, 1970). After migration, the proteins were visualized by a silver stain after oxidation with periodic acid (Pas dye) (Dubray et al, 1982).

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Electrophoretic actions were performed in the presence and absence of a reducing agent. The protein strips were further identified by a Western blot on nitrocellulose according to Towbin (towbin et al, 1979) and the evaluation was done with antibodies either directed against the gp 160 antigen or against host cells (CV-1) or proteins from the vaccinia virus.

(vi) Release of monoclonal antibody 178.1

The presence of monoclonal antibody 178.1 in the purified gp 160 antigen was measured by ELISA. The antibodies were captured by a goat-anti-mouse antibody and detected with a niotinylated anti-mouse antibody. Subsequent detection was performed with the streptavidin-peroxidase system.

(vii) Size exclusion chromatography of the gp 160 env gold purified gp 160 was chromatographed on a TSK 4000 SW HPLC column (7.5 mm x 300 mm) equilibrated in 0.2 M phosphate buffer pH 7 supplemented with 1% OGP. The flow rate was 0.75 ml / min and column fractions were analyzed for antigen by ELISA.

(viii) Determination of purity

Purity was determined after each purification step by SDS-polyacrylamide gel electrophoresis under reducing conditions. The protein bands were visualized by PAS staining and later identified by Western blot.

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The results clearly indicated that the antigen was free of contaminating proteins detectable after chromatography on the immunoaffinity column. However, the final product can be contaminated with trace amounts of monoclonal antibody, freeing itself from the immunoaffinity column. Thus, the amount of 178.1 antibody in the pure gp 160 was measured by ELISA. and varied between 0.004% and 0.02% of the total amount of protein present in the samples.

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The purified antigen was analyzed for the presence of oligomeric forms of gp 160. The formation of oligomers may reflect the structure of gp 160 in the virus where the envelope proteins are organized as spikes on the surface of the virion. The presence of cysteines in the primary sequence of the antigen allows the formation of (homo -) - oligomers linked by disulfide bonds. This was demonstrated by polyacrylamide gel electrophoresis in the presence and absence of β-mercaptoethanol. Without a reducing agent, the antigen revealed protein ranges of molecular mass greater than 160,000 Da, even in the presence of detergent (SDS). In contrast, when the antigen was boiled in the presence of a reducing agent, a single band of protein was observed at 160 kDa. In order to determine the size of the oligomeric structures, the antigen was analyzed by size exclusion HPLC chromatography. It was found from the calibration of the column with standards of known molecular mass that most of the 160 gp molecules were eluted in a retention time corresponding to a molecular mass of 640,000 Da. Thus, it was concluded that in the presence of a detergent but without a reducing agent, most of the HIV gp 160 env protein was assembled in tetrameric structures.

(ix) Analysis

The final product has an antigen / protein ratio of about 2 which corresponds to the ELISA content of the lysate (0.8-1.4 mg of antigen per ELISA per liter of culture).

Example 2

BHK-21 modified qp 160 expression and env, gp purification

160 from cells grown in suspension

The HIV wrapping protein gp 16.0 expressed in BHK-21 cells by a method analogous to that described in Example 1 (i) with a recombinant vaccinia virus was purified according to the scheme indicated in Example 1 (ii) except for some modifications less important in the lysis step.

Step 1: Lysis of BHK-21 cells and extraction of qp 160 „_ 9 antigen

After thawing, the cell suspension (10 cells) was centrifuged for 15 minutes at 2,200 xg and the floating product was discarded. The cell pill was resuspended in 50 ml of 30 mM Tris / HCl pH 8 buffer supplemented with 150 mM NaCl, 1% polyethylene glycol 300 monodecylether (Decyl PEG) and 20 mcg / ml aprotinin. The cells were lysed for 1 hour on ice with occasional manual agitation and centrifuged at 11,300 xg for 15 minutes. After separating the floating product, the pill was washed with 20 ml of lysis buffer and centrifuged at 11,300 xg for 15 minutes. The pill was eliminated and the combined floating products were used for further purification.

Steps 2 and 3 and subsequent analysis were performed as described in Example 1 (ii) - (ix).

Example 3

Preparation of Vaccine formulations gp 160 - 3D-MPL (a) rgp 160-Aluminum Hydroxide plus purified 3D-MPL rgp 160 (100 Mg or 20 μ $ each per dose) of vaccine (example 1) was adsorbed overnight at 4 ° C in aluminum hydroxide (alum) corresponding to 0.5 μg equivalents

3+ of Al in 1 ml of 150 mM NaCl, 10 mM phosphate buffer pH 6.8. After overnight incubation, the adjuvant preparation was centrifuged and the floating product was removed. An equal volume of adsorption buffer containing 100 μg of 3D-MPL was then added to rgp 160 bound to alum. It was found that more than 95% of rgp 160 was adsorbed on aluminum hydroxide.

(b) 160-3D-MPL rgp oil in water vehicle emulsion was prepared as follows. Pluronic L121 5% (BASF Wyandotte, New Jersey) (v / v) and 10% squalene (Aldrich) were added to phosphate buffered saline (PBS) containing 0.4% (v / v). of Tween 80. This mixture was then microfluidized. For microfluidization, the emulsion was cyclically displaced ten times through a microfluidizer (Model M110 Microfluidics Corp., Newton, Mass.). The resulting emulsion comprised only submicron particles. One volume of this emulsion was mixed with an equal volume of rgp 160 concentrated twice (or 20 μg or 100 μg) and vortexed in a short period to ensure complete mixing of the components. 100 µg / ml of 3D-MPL was then added to this 160 o / w rgp emulsion. The final preparation consisted of 0.2% Tween 80, 2.5% Pluronic L121, 5% squalene, 100 μg of 3D-MPL and rgp 160 (100 μg or 20 Mg) in an injection dose of 1 ml.

Example 4

Immunogenicity in guinea pigs

ELISA and neutralization titers

Five guinea pigs were immunized with 3 injections of 50 p, g of modified gp 160 (Example 1) in SAF-1 (Syntex adjuvant formulation-1) Byars NE and Allison A, C, (1987) Vaccine 5: 223-227 with an interval of one month. Sera were tested 2 weeks and 1 month after secondary immunization, as well as 2 weeks after tertiary immunization of guinea pigs. The results are described in Table 2.

A capture enzyme immunoassay (EIA) based on a lysate of HIV-1 infected cells (IIIB) was used to determine the ELISA titer of the antisera after the first and second boost. The test used is very similar to that published by Moore et al., [Moore J.P. et al., 1989, AIDS 3.155 (63)].

The microplate neutralization assay is based on the detection of HIV gag antigen in indicator cells. Briefly, SupTl cells (Hecht et al., 1984, Science 226: 1145) are used as indicator cells. The viral inoculum consists of a free floating product of a lymphoid cell line producing HIV-1 (IIIB). The floating product is centrifuged at a high speed to eliminate cells and cellular debris, divided into aliquots in 1 ml flasks and stored at -80 ° C until use. The sera to be tested are inactivated at 56 ° C for 30 minutes before testing. Our negative control consists of a collection of sera from animals pre-immunized or inoculated with adjuvant only (the same species as

sera to be tested). For neutralization, 750 TCID _5Q are incubated with double dilution series of sera for 1 hour at 37 ° C. SupTl cells are then added (4.10 ⁴ cells / well) and incubated 4 days at 37 ° C. The cytopathic effect is monitored microscopically, Triton X-100 (1% final concentration) is added to each container and the plate is frozen. A sandwich ELISA is used to monitor the relative amount of viral antigen produced in the cultures. The plates are coated with an anti-p55 monoclonal antibody. Previous samples treated with Triton X-100 are incubated on the plate and the presence of gag antigen is visualized by biotinylated HIV-1 + human IgGs following a step with streptavidin peroxidase. The percentages of reduction in HIV-1 antigen production compared to the control are then assessed for all tested serum dilutions. Using a curve adapted to the data points by nonlinear analysis of minimum squares, the dilution of the serum (if any) giving rise to a 50% reduction in antigen production compared to the control, is extrapolated.

Table 2 indicates the neutralizing antibody titer after tertiary immunization.

The neutralization titers observed after the second boost exceed those found in the sera of infected humans. More precisely, the titer of neutralization of our antisera in relation to the HIV IIIB isolate is on average 4 times higher than that observed for a group of 5 seropositive WHO reference sera (McKeating et al., 1989, J. Gen. Virol. 70.:3326-3333). The neutralization of a series of HIV-1 isolates (cross-neutralization) was tested by Dr. Weiss's laboratory (Chester Beatty Laboratories, U.K.) using a more rigorous neutralization test that provides a lower sensitivity and titer. The results, described in Table 3, were

reproduced on 3 occasions with 2 different blood samples and show good cross-neutralization of a series of HIV-1 strains including an African strain (CBL-4) (see Table 3).

Sera from guinea pigs immunized with vaccinia gp 160 (Example 1) (01-05) show good cross-neutralization titers after tertiary immunization.

Due to the strong cross-neutralization response observed after 3 doses, vaccinia gp 160 of the invention is considered to be of potential use for HIV-1 vaccination.

Example 5

Study of the effect of different vaccine formulations on the immunogenicity of purified HIV vaccinia recombinant gp 160 (isolate IIIB) in Rhesus monkeys

In this study, the ability of different vaccine formulations to improve the immunogenicity of purified vaccinia recombinant gp 160 (rgp 160) was evaluated in Rhesus monkeys (Macaca mulatta). The adjuvants tested were aluminum hydroxide (Alhydrogel, Superfos -Dennmark) in combination with 3D-MPL (Example 2b) (Monophosphoryl-lipid A 3D, Ribi); 3D-MPL in water emulsion (Example 2a).

Rhesus monkeys (Macaca mulatta) weighing 3.5 to 5 kg were randomly assigned to seven groups containing 3 or 4 animals per group.

The groups were immunized with different doses of rgp 160 stimulated in 3 adjuvant formulations, as follows:

Group 1 (4 monkeys): 100 pg rgp 160 absorbed in aluminum hydroxide plus 3D-MPL.

Group 2 (4 monkeys): 20 pg rgp 160 adsorbed on aluminum hydroxide plus 3D-MPL

Group 3 (4 monkeys): 100 pg rgp 160 plus 3D-MPL in o / w emulsion Group 4 (4 monkeys): 20pg rgp 160 plus 3D-MPL in o / w emulsion

5.1. Anti-Adjuvant Preparations

All rgp 160 formulations were prepared immediately before use. Each injection dose was administered in a volume of 1 ml.

Groups of Rhesus monkeys were injected intramuscularly into brachial triceps with a 1 ml dose of various formulations of gp 160 on day 0 and day 35. Two weeks after the second immunization, the animals were bled for antibody determinations.

5.2 Reading

Sera collected from these animals 2 weeks after the second injection were tested in ELISA and neutralization assays.

5.3 ELISA

A modification of the capture immunoassay was used whose general protocol described in Thiriart et al (Thiriart et al, 1989, J. Immunol 148 6.:832-1836). This assay uses a Biochrom commercial monospecific anti gp 41 reagent. A raw lysate of BHK 21 cells infected with vaccinia gp 160 is used as an antigen.

5.4 Neutralization Test

The microplate neutralization assay is based on the detection of HIV gag antigen in indicator cells. Briefly, SupTl cells (Hecht et al, 1984, Science 226: 1445) are used as indicator cells. The viral inoculum consists of a free floating product of cells of lymphoid cell line producing HIV-1 (IIIB). The floating product is centrifuged at high speed in order to eliminate cells and cellular debris, divided into aliquots in 1 ml flasks and stored at -80 ° C until use. The sera to be tested are inactivated at 56 ° C for 30 minutes before testing. The negative control consists of a pool of sera from pre-immunized animals. For neutralization, 750 TCID ₅₀ are incubated with double dilution sera for 1 hour at 37 ° C. Stup TI cells are then added (2.10 ⁴ cells / vessel) and incubated 4 days at 37 ° C. The cytopathic effect is monitored microscopically, Triton X-100 (1% final concentration) is added to each container and the plate is frozen. A sandwich ELISA is used to monitor the relative amount of viral antigen produced in the cultures. The plates are coated with an anti-p55 monoclonal antibody. Previous samples treated with Triton X-100 are incubated on the plate and the presence of gag antigen is visualized by biotinylated HIV-1 + human IgGs following the streptavidin peroxidase step. The percentages of reduction in HIV-1 antigen production relative to controls are then assessed for all tested serum dilutions. Using a curve adapted to the data points by linear regression analysis, the dilution of the serum (if any) giving rise to a 50% reduction in antigen production compared to controls, is extrapolated.

5.5 Results

Table 4 indicates the ELISA and neutralizing antibody titer after a second immunization. The general immunogenicity of HIV rgp 160 in 3D-MPL o / w is very good. When doses of 20 / xg of gp 160 are administered to the animals, the neutralization titer (NT) and the ELISA titer observed were extremely good in the group of animals receiving rgp 160 in 3G-MPL o / w (group 4). These data suggest a superior adjuvant effect of the 3D-MPL o / w emulsion. The immune response of animals immunized with gp 160 adsorbed on Alum in the presence of 3D-MPL is weak, although some neutralizing antibody is produced. HIV gp 160 contains a hydrophobic portion and this probably gives the molecule a high affinity for lipids. HIV gp 160 is best presented to the immune system using an oil in water based on the formulation containing 3D-MPL.

Example 6

6.1 Experimental scheme

Two chimpanzees (No. 1, No. 2) 160 purified vaccinia recombinant from example 1.

were immunized with gp (100 / xg / dose) (rgp 160)

Two chimpanzees (No. 3, No. 4) were immunized with purified recombinant gp 120 (100 / xg / dose) expressed in Drosophila Schneider cells (rgp 120) (Culp et al., Biotechnology, 9: 173-177, 1991 ).

The recombinant proteins were formulated with 3D MPL (100 / xg / dose) in an o / w emulsion. The formulation consisted of 0.2% Tween 80, 2.5% Pluronic L121, 5% squalene, 100 / xg 3D

MPL and recombinant antigen dose (100 μg for rgp 160 or rgp 120; in an injection volume of 1 ml.

The animals were immunized intramuseularly in one leg at ο, l and 2 months.

The animals were bled every two weeks for antibody determination by ELISA and neutralization assays (see Example 4.3 for ELISA methodology); the HIV neutralization assay has been slightly modified, as described below). The induction of delayed type hypersensitivity response (DTH) was also assessed, as described below.

6.2

Induction of humoral immunity

As indicated in Table 5, chimpanzees vaccinated with either rgp 160 or rgp 120 administered in a 3D MPL o / w emulsion produced high ELISA and neutralization titers after 3 immunizations. Neutralization activity could be detected in 3 sera out of 4 after 2 injections and in 4 out of 4 after another boost. An increase in neutralization titers was observed after a third immunization. The ELISA and neutralization titers measured after this boost are very similar to those obtained in rhesus monkeys immunized three times with the same formulation of rgp 160.

6.3 DTH induction

DTH tests were performed two weeks after the third immunization. All injections were administered in the abdomen, in a volume of 100 μΐ per injection.

The two chimpanzees immunized with rgp 160 and the two animals treated with rgp 120 underwent skin tests with 3 different antigens (rgp 160, rgD 26, tetanus toxoid) and 2 control buffers (rgDt control buffer (PBS) and control rgp 160). Different doses of recombinant antigen were tested: 40, 20 or 10p, g for 160 rgp and 20, 10 or 5 μ $ for rgDt.

DTH responses were monitored 24 hours later by measuring skin thickness. The results are illustrated in Figures 1-3.

A specific DTH response against tetanus toxoid and rgp 160 was observed in animals vaccinated with either rgp 160 (Fig. 1) or rgp 120 (Fig. 2).

These results indicate that formulations containing 3D MPL in an o / w emulsion are capable of inducing a specific T cell response (see Table 6).

In conclusion, the results obtained in chimpanzees clearly indicate that adjuvant formulations containing 3D MPL in an oil-in-water emulsion significantly improve the humoral (neutralizing antibodies) and effector cell-mediated (DTH) immune responses in primates.

Neutralization test

The microplate neutralization assay is based on the visual assessment of CPE induced by HIV1 infection in indicator cells. Briefly, SupTl cells (Hecht et al, 1984, Science 226: 1445) are used as indicator cells. The viral inoculum consists of a cell-free floating product from a lymphoid cell line producing HIV-1 (IIIB). The product

float is centrifuged at high speed in order to eliminate cells and cell debris, is divided into aliquots in 1 ml flasks and stored at -80 ° C until use. The sera to be tested are inactivated at 56 ° C for 30 minutes before testing. Our negative controls consist of a collection of sera from pre-immunized animals. For neutralization, 750 TCID _5q are incubated with a series of double dilutions of the sera for 1 hour at 37 ° C. SupTl cells are then added (2.10 ^ cells / well) and incubated 4 days at 37 ° C. The cytopathic effect is monitored microscopically on day 4 and neutralization titers are determined visually. The neutralization titers obtained correspond to the reciprocal of the serum dilution giving rise to an 80% reduction in the formation of syncytia in comparison with Preimmunization Controls. The visually determined titers are further objectified on day 7 by measuring cell viability in each container using the MTT Virol assay. Methods 20.:309-321, 1988).

assays represent the reciprocal of the serum dilution giving 80% protection against CPE compared to uninfected cells. The visually determined and MTT titers are very reproducible from one test to another and the MTT determined titers (not shown) are 2 to 4 times lower than those visually determined.

described by Pauwels et al (J. The titles determined in this

Table 1

Purification of HIV gp 160 expressed in CV-1 and BHK-21 cells

Step Purification Volume (ml) : Total Protein (mg) ^: Protein Recovery (%) Recovery Antigen (%) CV-1 (1) Lise 1400 2000 100 100 lentil lectin 350 100 5 80 Immunoaffinity 60 8 0.4 65 BHK-21 (2) Lise 1480 2688 100 100 lentil lectin 495 113 4 79 Immunoaffinity < . 60 7 0.3 58

(1) ; 20 1 culture as starting material (2): 14 1 culture (2.1 x 10 cells) as starting material

Table 2

IMMUNOGENICITY OF GP 160 IN COBAIAS

Anti-HIV Title (D Titule Neutrali _{Z a} ^ _IIB) Antigé ... ni-o- * Dose Adjuvant Guinea Post II Post III Post II Post III pig n ° 15 d 1 month 15 d 15 d 1 month Vaccine gP l60 50 ug SAF-1 01 02 21653 22348 20327 16694 158589 179971 183 165 <100 117 2207 1930 (Example 1) 03 25360 20327 151254 445 394 6630 D 04 35905 26175 161124 353 195 2034 05 ND 8658 77884 133 <100 677 GMT 25764 17330 140203 229 117 2079

(1) Represents the reverse of the dilution of the serum that gives an O.D. of 0.5

Table 3

Heterologous challenge SAMPLE I.D. Illb Homolo- gous challenge SF2 MN RF CBL-4 01 02/05/90 80 10 / <10 - 02 5/2/90 <40 80 10 - 10 03 5/2/90 80 20 - - 10 04 5/2/90 40 10 - - 10 05/05/90 80 40 10 - 10 01 05/14/90 40 20 - 10 02 5/14/90 80 20 - - 10 03 5/14/90 160 20 10 - 10 / <10 04 05/14/90 20 10 - - 40 05/05/14/90 40 40 0 / <10 - 10 1 Positive Human 160 320 80 160 80 Human Negative - - - - -

° Neutralizing activity of Sera da Cobaia for gp 160 'in relation to homologous isolates (Illb) and heterologues.

Positive neutralizing effect is represented by 90% inhibition of syncytium formation as assessed visually.

Table 4

IMMUNOGENICITY OF GP 160 OF RECOMBINANT VACCINIA IN RHESUS MONKEYS

Γ -. __ ______ TELISA and Neutralization of Titles 1 "· -15 days ....... ___..... _____ E.os.1 — U— Ί Group ADJ / DOSE Identification 50% NT * ELISA ** ._____ point title central 1 ALUM 3D IP 111 <100 576 MPL / 100gg IP 123 <100 301 IP 151 <100 411 IP 167 <100 630 GMT 460 2 ALUM 3D IP 101 <100 1871 MPL / 20gg IP 103 <100 1641 IP 105 <100 1437 IP 196 <100 94 GMT 802 3 3D MPLOW IP 120 486 9043 lOOgg IP 125 282 5537 IP 150 285 3981 IP 152 918 10629 GMT 435 6789 4 · 3D MPLOW IP 114 462 5413 20gg IP 117 576 5220 IP 118 <100 2529 IP 197 804 5268 ‘ GMT 322 4404

* Corresponding to the reciprocal of the serum dilution giving rise to

50% reduction in antigen production compared to controls.

** Corresponds to the reciprocal of the dilution of the serum giving rise to an absorption equal to 50% of the maximum absorption value (title of central point)

GMT = Geometric Average Title

Table 5

ANTI-HIV ANTIBODY RESPONSE IN VACCINATED CHIMPANZES WITH RPG 160 AND RPG 120

Post I Post II Post III 20 days 14 days 20 days 14 days 20 days Chimp vaccine Tit. Neu t. Tit. ELISA Tit. Neut. Tit.Neut. ' Elisa Tit. nt. Neut. Tit. Neut. rgpl60 (100gg) 1 <50 2592 <50 / 50 / 16509 200 400 3D MPL o / w 2 <50 9004 100 100 15665 200 / 400 rgpl20 (100 | lg) 3 <50 3059 100 100 22679 000 000 3D MPL o / w 4 <50 506 <50 / 50 3917 200 200 /

ELISA TITLE: corresponds to the reciprocal of the dilution of the serum giving rise to an absorption equal to 50% of the maximum absorption value (central point title).

TITLE NEUT .: corresponds to the reciprocal of the serum dilution giving rise to 80% protection from the cytopathic effect of isolate IIIB

HIV1.

/: bar indicates that the neutralization titre is between 2 successive dilutions of the serum; for example,. 200 / indicates that the title is between 200 and 400.

Table 6

ANTI-HSV ANTIBODY RESPONSE IN VACCINATED CHIMPANZES

Post II Post III 14 days 20 days 14 days days Vaccine Chimp. Title Elisa Title Neut. Title Elisa Title Neut. Title Title Elisa Neut. Title Title Elisa Neut. Name rgplGO (100gg) 3D MPL o / w 1 <100 <50

>

ELISA TITLE: corresponds to the reciprocal of the dilution of the serum giving rise to an absorption equal to 50% of the maximum absorption value (title of central point).

TITLE NEUT .: corresponds to the reciprocal dilution of the serum giving rise to 100% protection against the cytopathic effect of HSV2

Claims (3)

CLAIMS: was going. - process for the production of a modified HIV gplôO protein in which amino acids other than lysine or arginine are provided at positions 502 and 510 independently, characterized by comprising the expression of a DNA sequence encoding said protein in a recombinant eukaryotic host. 2§. Process according to claim 1, characterized in that the amino acids at position 502 and 510 are selected from the group: histidine, threonine, serine, asparagine, aspartic acid, glutamine and glutamic acid. 32. The method of claim 1 or 2, characterized in that the amino acids at positions 502 and 510 are glutamic acid. 42. - Process for the production of an oligomer or dimer of a modified gpl60 in which amino acids other than lysine or arginine are provided at positions 502 and 510 independently, characterized by comprising the isolation of the modified protein from a recombinant host and its purification in non-reducing conditions. 52. - Method for the production of a vaccine or pharmaceutical composition, characterized by comprising the mixture of a modified gpl60 in which amino acids other than lysine or arginine are provided in position 502 and 510 independently, or an oligomeric form thereof, with a pharmaceutically acceptable vehicle. 6§. - Method for producing a vaccine or pharmaceutical composition, characterized by comprising a mixture of gpl60 or an immunological derivative thereof, and monophosphoryl-lipid A 3D, with a pharmaceutically acceptable carrier.